NOX2 is the primary source of superoxide in the macula densa in angiotension II induced hypertension

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Tubuloglomerular feedback (TGF) is one of the main mechanisms that regulate the renal microcirculation, NaCl excretion, and ultimately blood pressure. Superoxide (O−2) in the macula densa (MD) enhances TGF by scavenging nitric oxide. MD expresses NOX2 and NOX4 isoforms of NAD(P)H oxidase. We hypothesize that NOX2 is the primary isoform for O−2 in angiotensin II (Ang II) induced hypertension.MD like cell line, MMDD1, was used. O−2 was measured using lucigenin chemiluminescence with a luminometer and expressed by units/min/106 cells.To measure whether Ang II induces O−2 production in MMDD1 cells, we stimulated the cells with Ang II (106 M) for 30 min. O−2 production increased from 198.4 ± 14.7 to 447.6 ± 21.2 units/min/105 cells. To determine if the Ang II induced O−2 is mediated through AT1 or AT2 receptors, we used specific receptor antagonists. In the presence of AT1 receptor antagonist losorten (10−6 M), O−2 was from 198.4 ± 14.7 to 214.7 ± 35.4 unit/min/106 cells. In the presence of AT2 receptor antagonist PD‐123319 (10−6 M), O−2 was from 198.4 ± 14.7 to 435.1 ± 25.3 unit/min/105 cells. To study which NOX is the primary source of Ang II induced O−2 , we knocked down NOX2 or NOX4 with siRNA and measured O−2. In the cells treated with siRNA‐NOX2, Ang II induced O−2 was from 163.9 ± 32.6 to 239.5 ± 51.8 unit/min/105 cells. In the cells treated with siRNA‐NOX4, O−2 was from 117.2 ± 19.3 to 422.8 ± 21.7 unit/min/105 cells. Scramble treated cells are not different in O−2 when compared with control.ConclusionsAng II induces O−2 production in MMDD1 cells mediated via AT1 receptors. NOX2 is the primary source of in Ang II induced O−2.